Dibutyril camp

hNSC were dissociated into single cells using StemPro accutase cell dissociation reagent (Life Technologies) and further suspended in the solution containing laminin-functionalized PEG-4MAL precursors (4 × 10⁶ viable cells/mL). Cell-laden hydrogels were formed by mixing the PEG-4MAL precursor solution containing cells with the MMP2-sensitive peptide and PEG-dithiol cross-linkers, dissolved in 10 mM HEPES in PBS (pH 6.5), at different molar ratios (%), and subsequently incubating the polymerizing gels at 37 °C, 5% CO₂ for 15 minutes. hNSC were initially cultured in expansion medium and then induced to differentiate along the neuronal lineage by growth factor withdrawal. Briefly, at day 2 of culture, the medium was switched to the StemPro NSC SFM media-Neurobasal/B27 (Life Technologies) (1:1) mix, without growth factors. At day 8, half of the medium was replaced by the StemPro NSC SFM-Neurobasal/B27 (1:3) mix supplemented with 10 ng/mL of brain-derived neurotrophic factor (BDNF; Peprotech) and 500 μM of N⁶, 2’-O-Dibutyryladenosine 3’, 5’-cyclic monophosphate sodium salt (dibutyril cAMP; Sigma). Half of the medium was changed every other day, up to 14 days. hNSC cultured within unmodified and PEG-4MAL gels containing entrapped msLn-111 (100 μg/mL) were herein used as controls.